Digital microfluidics apparatuses and methods for manipulating and processing encapsulated droplets

ABSTRACT

Air-matrix digital microfluidics (DMF) apparatuses and methods of using them to prevent or limit evaporation and surface fouling of the DMF apparatus. In particular, described herein are air-matrix DMF apparatuses and methods of using them including thermally controllable regions with a wax material that may be used to selectively encapsulate a reaction droplet in the air gap of the apparatus; additional aqueous droplets may be combined with the encapsulated droplet even after separating from the wax, despite residual wax coating, by merging with an aqueous droplet having a coating of a secondary material (e.g., an oil or other hydrophobic material) that may remove the wax from the droplet and/or allow combining of the droplets.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application claims priority to U.S. provisional patentapplications No. 62/481,488, titled “DIGITAL MICROFLUIDICS APPARATUSESAND METHODS FOR MANIPULATING AND PROCESSING ENCAPSULATED DROPLETS,” andfiled on Apr. 4, 2017; U.S. provisional patent application No.62/553,743, titled “DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USINGTHEM,” field on Sep. 1, 2017; and U.S. provisional patent applicationNo. 62/557,714, titled “DIGITAL MICROFLUIDICS DEVICES AND METHODS OFUSING THEM,” filed on Sep. 12, 2017, each of which is hereinincorporated by reference in its entirety.

This patent application may be related to U.S. patent application Ser.No. 15/579,455, titled “AIR-MATRIX DIGITAL MICROFLUIDICS APPARATUSES ANDMETHODS FOR LIMITING EVAPORATION AND SURFACE FOULING,” filed on Jun. 6,2016, which claimed priority to U.S. Provisional Application 62/171,756entitled, “DEVICE AND METHODS FOR LIMITING EVAPORATION AND SURFACEFOULING,” filed on Jun. 5, 2015, which is herein incorporated byreference in its entirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

FIELD

Air-matrix digital microfluidic (DMF) apparatuses and methods formanipulating and processing encapsulated droplets are described herein.

BACKGROUND

Microfluidics has transformed the way traditional procedures inmolecular biology, medical diagnostics, and drug discovery areperformed. Lab-on-a-chip and biochip type devices have drawn muchinterest in both scientific research applications as well as potentiallyfor point-of-care applications because they carryout highly repetitivereaction steps with a small reaction volume, saving both materials andtime. While traditional biochip-type devices utilize micro- ornano-sized channels and typically require corresponding micropumps,microvalves, and microchannels coupled to the biochip to manipulate thereaction steps, these additional components greatly increase cost andcomplexity of the microfluidic device.

Digital microfluidics (DMF) has emerged as a powerful preparativetechnique for a broad range of biological and chemical applications. DMFenables real-time, precise, and highly flexible control over multiplesamples and reagents, including solids, liquids, and even harshchemicals, without need for pumps, valves, or complex arrays of tubing.In DMF, discrete droplets of nanoliter to microliter volumes aredispensed from onto a planar surface coated with a hydrophobicinsulator, where they are manipulated (transported, split, merged,mixed) by applying a series of electrical potentials to an embeddedarray of electrodes. Complex reaction steps can be carried out using DMFalone, or using hybrid systems in which DMF is integrated withchannel-based microfluidics.

Despite significant advances, both evaporation, particularly inair-matrix DMF, and surface fouling remains issues. Surface foulingoccur when components from the reaction mixture irreversibly adheres tosurfaces of the microfluidic or DMF device after contacting thesesurfaces. Surface fouling is a particularly acute problem when operatinga higher (e.g., greater than 37° C.) temperatures. Various strategieshave been proposed to prevent surface fouling, such as using polymers,glass, and metals to fabricate the microfluidic channels or chemicalmodification of material surfaces. However, these strategies have hadlimited success, particularly in the context of DMF, despite efforts totest and fabricate surfaces and surface coatings that are resistant tosurface fouling. In some instances, a coating intended to preventsurface fouling may cause undesirable interactions and secondaryreactions with the reaction mixture and/or reagents used. In general, itwould be desirable to have a simple solution to minimizing surfacefouling in microfluidic and DMF devices.

Evaporation is also a concern when performing reactions in an air-matrixDMF device. In general, an air-matrix DMF apparatus may refer to anynon-liquid interface of the DMF apparatus in which the liquid dropletbeing manipulated by the DMF apparatus is surrounded by an air (or anyother gas) matrix. As used herein, an air-matrix may also andinterchangeably be referred to as a “gas-matrix” DMF apparatus; the gasdoes not have to be air, though it may be. Evaporation may be especiallyproblematic in air-matrix DMF methods and that heat for a prolongedperiod of time (e.g., greater than 30 seconds). Evaporation limits theutility of air-matrix DMF, because enzymatic reactions are often highlysensitive to changes in reactant concentration. Largely for this reason,others have attempted to use oil-matrix DMF for biochemicalapplications, despite numerous drawbacks including: the added complexityof incorporating gaskets or fabricated structures to contain the oil;unwanted liquid-liquid extraction of reactants into the surrounding oil;incompatibility with oil-miscible liquids (e.g., organic solvents suchas alcohols); and efficient dissipation of heat, which undermineslocalized heating and often confounds temperature-sensitive reactions.Another strategy for addressing evaporation has been to place theair-matrix DMF device in a closed humidified chamber, but this oftenresults in unwanted condensation on the DMF surface, difficult and/orlimited access to the device, and a need for additional laboratory spaceand infrastructure.

It has also been proposed to address evaporation by transferringreaction droplets from the air-matrix DMF device to microcapillaries,where they can be heated in dedicated off-chip modules withoutevaporation problems. However, this complicates design and manufactureof the air-matrix DMF device, and introduces the added complications ofmicrocapillary interfaces and coordination with peripheral modules.

Thus, there exists a need for air-matrix DMF apparatuses and methodsthat may prevent or limit evaporation and/or prevent or limit surfacefouling. Described herein are apparatuses and methods that may addressthis need.

SUMMARY OF THE DISCLOSURE

A typical DMF apparatus may include parallel plates separated by an airgap; one of the plates (typically the bottom plate) may contain apatterned array of individually controllable actuation electrodes, andthe opposite plate (e.g., the top plate) may include one or more groundelectrode. Alternatively, the one or more ground electrode(s) can beprovided on the same plate as the actuating (e.g., high-voltage)electrodes. The surfaces of the plates in the air gap may include ahydrophobic material which may be dielectric or in some variations anadditional dielectric layer may be included. The hydrophobic and/ordielectric layer(s) may decrease the wettability of the surface and addcapacitance between the droplet and the control electrode. Droplets maybe moved or otherwise manipulated while in the air gap space between theplates. The air gap may be divided up into regions, and some regions ofthe plates may include heating/cooling by a thermal regulator (e.g., aPeltier device, a resistive heating device, a convective heating/coolingdevice, etc.) that is in thermal contact with the region, and may belocalized to that region. Reactions performed on with the air-matrix DMFapparatus may be detected, including imaging or other sensor-baseddetection, and may be performed at one or more localized regions or overall or over a majority of the air gap space of the air-matrix DMFapparatus.

Any of the apparatus variations described herein may include wax withinthe reaction chamber. As described herein, wax may be included in theair gap even if a separate reaction chamber apparatus is included. Thewax may be present in a thermal zone (e.g., a thermally controlledsub-region of the air gap) as a solid (e.g., a wall, channel, cave, orother structure of wax) that can be melted to form a liquid and combinedwith a reaction droplet. The liquid wax, upon mixing together with thereaction droplet, will typically form a coating over and around theliquid droplet, protecting it from evaporation.

Unfortunately, following the use of wax to encapsulate a droplet in anair gap to prevent evaporation, in some cases it may be difficult tofully separate the wax material from the aqueous droplet. Thus, addingadditional aqueous droplets may be difficult or impossible, as the smallamount of residual wax material may form an outer coating of wax thatwill prevent merging of the coated droplet with other aqueous droplets.Thus, described herein are methods and apparatuses for performing one ormore droplet operations (including merger with additional aqueousdroplets) on an at least partially outer wax-coated aqueous droplet.

Any of the methods described herein may be referred to as a method ofperforming droplet operations on a droplet that is at least partiallycoated in wax and within an air-matrix digital microfluidic (DMF)apparatus (e.g., within the air gap of the air-matrix DMF apparatus). Ingeneral, these methods may include starting with the aqueous droplet(e.g., adding it into the air gap of the DMF apparatus), and usingelectrowetting to move it within the air gap. The methods may alsogenerally include encapsulating with a wax (wax material) as describedin more detail. This may be used to perform a thermally-regulatedprocedure. Further, any of these methods may also typically includeremoving much or most of the wax from the aqueous droplet, e.g., byreducing the temperature to solidify the wax while moving the dropletaway (e.g., by electrowetting).

In addition, any of these methods may generally include: moving, byelectrowetting, an aqueous reaction droplet having an outer coating ofwax (which may be just a very thin layer at least partially coating theaqueous droplet) within an air gap of the air-matrix DMF apparatus (asexpected, the air gap may be formed between a first plate and a secondplate of the air-matrix DMF apparatus). Any of these methods may alsoinclude merging the aqueous reaction droplet with a carrier dropletcomprising an aqueous droplet coated with an oil or an organic solventin the air gap to form a combined droplet. The oil or organic solventmay be coated in a thin (e.g., monolayer or thicker) layer on the secondaqueous droplet but interact with the wax coating on the first aqueousdroplet and permit the two to merge; in the absence of this oil ororganic solvent, the two droplets will not merge. Thereafter, themethods may include moving, by electrowetting, the combined dropletwithin the air gap. The second aqueous droplet may include any material(buffer, marker, beads, wash, etc.). This process may be repeatedmultiple times, e.g., by combining the combined droplet with additionalaqueous droplets including an oil or organic solvent.

In some variations, the oil or organic solvent material may be separatedfrom the combined droplet or target molecules in the combined droplet.For example, beads (e.g., magnetic beads) holding the target molecule(s)may be separated magnetically from the combined droplet, including theouter coating (e.g., wax and oil/organic solvent). The beads may then bewashed to remove any residual coating. Alternatively, in somevariations, the coating may be mechanically removed.

Described herein are air-matrix DMF apparatuses that include a waxmaterial in a solid state at room temperature and below, but mayselectively and controllably combined with a reaction droplet within theair gap when the wax structure is heated. For example, described hereinare air-matrix digital microfluidic (DMF) apparatuses configured toprevent evaporation. The apparatus may include a first plate having afirst hydrophobic layer; a second plate having a second hydrophobiclayer; an air gap formed between the first and second hydrophobiclayers; a plurality of actuation electrodes adjacent to the firsthydrophobic layer, wherein each actuation electrode defines a unit cellwithin the air gap; one or more ground electrodes adjacent to actuationelectrode of the plurality of actuation electrodes; a thermal regulatorarranged to heat a thermal zone portion of the air gap wherein aplurality of unit cells are adjacent to the thermal zone; a wax bodywithin the thermal zone of the air gap; and a controller configured toregulate the temperature of the thermal zone to melt the wax body and toapply energy to actuation electrodes of the plurality of actuationelectrode to move a droplet through the air gap.

The wax body may span one or more (e.g., a plurality of adjacent) unitcells. The wax body may comprise a wall of wax within the air gap. Insome variations the wax body forms a channel or vessel within the airgap. For example, the wax body may form a concave shape in the air gap,which may help it combine with a reaction droplet when heated. Ingeneral, the wax body may be melted immediately before combining withthe reaction droplet. In some variations the wax body may itself be adroplet (wax droplet) that is moved into position by the air-matrix DMFapparatus so that it can combine with the reaction droplet.

The wax body may be formed of any appropriate wax that is typicallysolid at room temperature, such as, e.g., paraffin wax. Other waxes maygenerally include hydrophobic, malleable solids near ambienttemperatures such as higher alkanes and lipids, typically with meltingpoints above about 40° C. (104° F.), that may melt to give low viscosityliquids. Examples of waxes include natural waxes (beeswax, plant waxes,petroleum waxes, etc.).

Any of these apparatuses may include features such as those describedabove, e.g., at least one temperature sensor in thermal communicationwith the thermal regulator. The plurality of actuation electrodes mayform a portion of the first plate. The one or more ground electrodes maybe adjacent to the second hydrophobic layer, across the air gap from thefirst plate. The apparatus may also include a dielectric between thefirst hydrophobic layer and the plurality of actuation electrodes (or insome variations the dielectric layer is the hydrophobic layer, as somehydrophobic layers are also dielectric materials). As mentioned above, athermal regulator may be a thermoelectric heater.

Also described herein are methods of preventing droplet evaporationwithin an air-matrix digital microfluidic (DMF) apparatus, the methodmay include: introducing a reaction droplet into an air gap of theair-matrix DMF apparatus which is formed between a first plate and asecond plate of the air-matrix DMF apparatus; melting a wax body withinthe air gap of the air-matrix DMF; combining the reaction droplet withthe melted wax body to protect the reaction droplet from evaporation;and allowing a reaction to proceed within the reaction droplet.

Melting the wax body typically comprises increasing the temperature of aportion of the air gap comprising a thermal zone to a temperature abovethe melting point of the wax forming the wax body. In some variations,melting the wax body comprises melting a solid wax body formed into awall or open chamber within the air gap.

Introducing the reaction droplet into an air gap may comprise combingmultiple droplets to form a reaction droplet within the air gap. Thefirst plate may comprise a plurality of adjacent actuation electrodes,and wherein combing the reaction droplet with the melted wax bodycomprises applying energy to a subset of the actuation electrodes of theplurality of adjacent actuation electrodes to move the reaction dropletin contact with the wax body prior to melting the wax body.

The first plate may comprise a plurality of adjacent actuationelectrodes, wherein combing the reaction droplet with the melted waxbody may comprise applying energy to a subset of the actuationelectrodes of the plurality of adjacent actuation electrodes to move thereaction droplet in contact with the melted wax body.

Allowing a reaction to proceed may comprise heating portion of the airgap containing the reaction droplet. As mentioned, any of these methodsmay include detecting a product within the reaction droplet.

Although the majority of the devices described herein are air-matrix DMFapparatuses that include two parallel pates forming the air gap, any ofthe techniques (methods and apparatuses) may be adapted for operation aspart of a one-plate air-matrix DMF apparatus. In this case, theapparatus includes a single plate and may be open to the air above thesingle (e.g., first) plate; the “air gap” may correspond to the regionabove the plate in which one or more droplet may travel while on thesingle plate. The ground electrode(s) may be positioned adjacent to(e.g., next to) each actuation electrode, e.g., in, on, or below thesingle plate. The plate may be coated with the hydrophobic layer (and anadditional dielectric layer maybe positioned between the hydrophobiclayer and the dielectric layer, or the same layer may be both dielectricand hydrophobic). The methods and apparatuses for correcting forevaporation may be particularly well suited for such single-plateair-matrix DMF apparatuses.

In some embodiments, an air-matrix digital microfluidic (DMF) apparatusconfigured to prevent evaporation is provided. The apparatus includes afirst plate having a first hydrophobic layer; a second plate having asecond hydrophobic layer; and an air gap formed between the first andsecond hydrophobic layers. The apparatus further includes a plurality ofactuation electrodes adjacent to the first hydrophobic layer; a thermalregulator arranged to heat a portion of the air gap configured as athermal zone; a wax body within the thermal zone of the air gap; and acontroller. The controller is programmed to actuate the plurality ofactuation electrodes to transport an aqueous reaction droplet throughthe air gap to the thermal zone; regulate the temperature of the thermalzone to melt the wax body into a liquid wax that encapsulates theaqueous reaction droplet; regulate the temperature of the thermal zoneto perform a reaction protocol within the liquid wax encapsulatedaqueous reaction droplet; actuate the plurality of actuation electrodesto transport the aqueous reaction droplet away from the thermal zone;actuate the plurality of actuation electrodes to bring a carrier dropletcomprising an oil or an organic solvent coated aqueous droplet to theaqueous reaction droplet; and merge the carrier droplet with the aqueousreaction droplet.

In some embodiments, the wax body spans a plurality of adjacentactuation electrodes of the plurality of actuation electrodes. In someembodiments, the wax body comprises a wall of wax within the air gap. Insome embodiments, the wax body forms a channel or vessel within the airgap. In some embodiments, the wax body comprises paraffin wax.

In some embodiments, the apparatus further includes at least onetemperature sensor in thermal communication with the thermal regulator.

In some embodiments, the plurality of actuation electrodes form aportion of the first plate.

In some embodiments, the thermal regulator comprises a thermoelectricheater.

In some embodiments, the carrier droplet comprises beads. In someembodiments, the beads are magnetic. In some embodiments, the beads areconfigured to bind to a molecule selected from the group consisting ofDNA, RNA, and proteins.

In some embodiments, the carrier droplet comprises a reagent, a primer,a dilution buffer, an enzyme, a protein, a nanopore, a wash buffer, analcohol, formamide, or a detergent.

In some embodiments, the wax body is disposed on a removable sheet thatcan be removably attached to either the first plate or the second plate.

In some embodiments, the first plate or the second plate are part of aremovable cartridge.

In some embodiments, a method of preventing droplet evaporation withinan air-matrix digital microfluidic (DMF) apparatus is provided. Themethod includes introducing an aqueous reaction droplet into an air gapof the air-matrix DMF apparatus which is formed between a first plateand a second plate of the air-matrix DMF apparatus; transporting theaqueous reaction droplet to a thermal zone of the air gap, the thermalzone comprising a wax; melting the wax within the thermal zone toencapsulate the aqueous reaction droplet with the wax; regulating thetemperature of the encapsulated reaction droplet to allow a reaction toproceed within the aqueous reaction droplet; transporting the aqueousreaction droplet away from the thermal zone after the reaction iscompleted; introducing a carrier droplet comprising an oil or an organicsolvent coated aqueous droplet into the air gap; and merging the aqueousreaction droplet with the carrier droplet to form a merged droplet.

In some embodiments, melting the wax comprises melting a solid waxformed into a wall or open chamber within the air gap.

In some embodiments, introducing the aqueous reaction droplet into anair gap comprises combining multiple droplets to form the aqueousreaction droplet within the air gap.

In some embodiments, the first plate comprises a plurality of adjacentactuation electrodes, and wherein combining the aqueous reaction dropletwith the melted wax comprises applying energy to a subset of theactuation electrodes of the plurality of adjacent actuation electrodesto move the aqueous reaction droplet in contact with the wax prior tomelting the wax.

In some embodiments, the first plate comprises a plurality of adjacentactuation electrodes, and wherein combining the aqueous reaction dropletwith the melted wax comprises applying energy to a subset of theactuation electrodes of the plurality of adjacent actuation electrodesto move the aqueous reaction droplet in contact with the melted waxafter melting the wax.

In some embodiments, the method further includes detecting a productwithin the aqueous reaction droplet.

In some embodiments, the method further includes mixing the reactiondroplet with a plurality of beads after the carrier droplet has beenmerged with the aqueous reaction droplet.

In some embodiments, the method further includes immobilizing the beadsafter the carrier droplet has been merged with the aqueous reactiondroplet.

In some embodiments, the method further includes moving the mergedcarrier droplet and aqueous reaction droplet away from the immobilizedbeads.

In some embodiments, the method further includes re-suspending theimmobilized beads with an aqueous droplet.

In some embodiments, the method further includes separating the beadsfrom the merged carrier droplet and aqueous reaction droplet by moving amagnet away from the merged carrier droplet and aqueous reactiondroplet.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is a top view of an example of a portion of an air-matrix DMFapparatus, showing a plurality of unit cells (defined by the underlyingactuating electrodes) and reaction chamber openings (access holes).

FIG. 2A shows the top view of FIG. 1 and FIGS. 2B-2D show side views ofvariations of reaction chamber wells that may be used in an air-matrixDMF apparatus. In FIG. 2B the reaction chamber well comprises acentrifuge tube; in FIG. 2C the reaction chamber well comprises a wellplate (which may be part of a multi-well plate); and in FIG. 2D thereaction chamber well is formed as part of the pate of the air-matrixDMF apparatus.

FIGS. 3A-3E illustrate movement (e.g., controlled by a controller of anair-matrix DMF apparatus) into and then out of a reaction chamber, asdescribed herein. In this example, the reaction chamber well is shown ina side view of the air-matrix DMF apparatus and the reaction chamber isintegrally formed into a plate (e.g., a first or lower plate) of theair-matrix DMF apparatus which includes actuation electrodes (reactionwell actuation electrodes) therein.

FIG. 4A shows a time series of photos of an air matrix DMF apparatusincluding a wax (in this example, paraffin) body which is melted andcovers a reaction droplet.

FIG. 4B is an example of a time series similar to that shown in FIGS.4A(3) and 4A(4), without using a wax body to cover the reaction droplet,showing significant evaporation.

FIG. 5 is a graph comparing an amplification reaction by LAMP with andwithout a wax covering as described herein, protecting the reactiondroplet from evaporation.

FIG. 6A show graphical results of LAMP using paraffin-mediated methods;this may be qualitatively compared to the graph of FIG. 6B showsgraphical results of LAMP using conventional methods.

FIGS. 7A and 7B show the encapsulation of a droplet within wax in athermal zone and the subsequent separation of the droplet from theliquid wax.

FIGS. 8A-8C show the merging of a carrier droplet with beads with thedroplet from FIGS. 7A and 7B and the subsequent separation andre-suspension of the beads.

DETAILED DESCRIPTION

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like.

Described herein are air-matrix digital microfluidics (DMF) methods andapparatuses that may minimize the effect of surface fouling and/orevaporation. An air-matrix DMF apparatus as described herein may beparticularly useful when heating the reaction droplets being processed.

In general, an air-matrix DMF apparatus as disclosed herein may have anyappropriate shape or size. As used herein, the term “surface fouling”may refer to accumulation of unwanted materials on solid surfaces,including with the air gap of the air matrix DMF apparatus (e.g., upperand/or lower plate surfaces). Surface fouling materials can consist ofeither living organisms (biofouling) or a non-living substance(inorganic or organic). Surface fouling is usually distinguished fromother surface-growth phenomena in that it occurs on a surface of acomponent, or system and that the fouling process impedes or interfereswith function.

The air-matrix DMF apparatuses described herein generally includes atleast one hydrophobic surface and a plurality of activation electrodesadjacent to the surface; either the hydrophobic surface may also be adielectric material or an additional dielectric material/layer may bepositioned between the actuation electrodes and the hydrophobic surface.For example, in some variations, the air-matrix DMF includes a series oflayers on a printed circuit board (PCB) forming a first or bottom plate.The outer (top) surface of this plate is the hydrophobic layer. Abovethis layer is the air gap (air gap region) along which a reactiondroplet may be manipulated. In some variations a second plate may bepositioned opposite from the first plate, forming the air gap regionbetween the two. The second plate may also include a hydrophobic coatingand in some variations may also include a ground electrode or multipleground electrodes opposite the actuation electrodes. The actuationelectrodes may be configured for moving droplets from one region toanother within the DMF device, and may be electrically coupled to acontroller (e.g., control circuitry) for applying energy to drivemovement of the droplets in the air gap. As mentioned, this plate mayalso include a dielectric layer for increasing the capacitance betweenthe reaction droplet and the actuation electrodes. The reaction startingmaterials and reagents, as well as additional additive reagents may bein reservoirs that may be dispensed into the air gap, where the reactionmixture is typically held during the reaction. In some instances thestarting materials, reagents, and components needed in subsequent stepsmay be stored in separate areas of the air gap layer such that theirproximity from each other prevents them from prematurely mixing witheach other. In other instances, the air gap layer may include featuresthat are able to compartmentalize different reaction mixtures such thatthey may be close in proximity to each other but separated by a physicalbarrier. In general, the floor of the air gap is in the first plate, andis in electrical contact with a series of actuation electrodes.

The air gap DMF apparatuses described herein may also include otherelements for providing the needed reaction conditions. For instance, theair gap DMF apparatuses may include one or more thermal regulators(e.g., heating or cooling element such as thermoelectric modules) forheating and cooling all or a region (thermal zone) of the air gap. Inother instances, heating or cooling may be provided by controllingendothermic or exothermic reactions to regulate temperature. The air gapDMF apparatuses may also include temperature detectors (e.g., resistivetemperature detector) for monitoring the temperature during a reactionrun. In addition, the DMF apparatuses may also include one or moremagnets that can be used to manipulate magnetic beads in an on demandfashion. For example, the magnet(s) can be an electromagnet that iscontrolled by a controller to generate a magnetic field that can agitateor immobilize magnetic beads.

Thus, the air gap DMF apparatuses described herein may include one ormore thermal zones. Thermal zones are regions on the air gap DMFapparatuses (e.g., the air gap) that may be heated or cooled, where thethermal zones may transfer the heating or cooling to a droplet withinthe thermal zone through one or more surfaces in contact with the airgap region in the zone (e.g., the first plate). Heating and cooling maybe through a thermal regulator such as a thermoelectric module or othertype of temperature-modulating component. The temperature of one or manythermal zones may be monitored through a temperature detector or sensor,where the temperature information may be communicated to a computer orother telecommunication device. The temperature is typically regulatedbetween 4° C. and 100° C., as when these apparatuses are configured toperform one or more reactions such as, but not limited to: nucleic acidamplifications, like LAMP, PCR, molecular assays, cDNA synthesis,organic synthesis, etc.

An air gap DMF apparatus may also include one or more thermal voids.Thermal voids may be disposed adjacent to the different thermal zones.The thermal voids are typically regions in which heat conduction islimited, e.g., by removing part of the plate (e.g., first plate)(forming the “void”). These voids may be strategically placed to isolateone thermal zone from another which allows the correct temperatures tobe maintained within each thermal zone.

In general, any of the air-matrix DMF apparatuses described herein mayinclude a separate reaction chamber that is separate or separable fromthe air gap of the apparatus, but may be accessed through the air gapregion. The reaction chamber typically includes a reaction chamberopening that is continuous with the lower surface of the air gap (e.g.,the first plate), and a reaction chamber well that forms a cup-likeregion in which a droplet may be controllably placed (and in somevariations, removed) by the apparatus to perform a reaction whencovered. The cover may be a mechanical cover (e.g., a cover the seals orpartially seals the reaction chamber opening, or a cover thatencapsulates, encloses or otherwise surrounds the reaction droplet, suchas an oil or wax material that mixes with (then separates from andsurrounds) the reaction droplet when the two are combined in thereaction chamber.

In general, the reaction chamber opening may be any shape or size (e.g.,round, square, rectangular, hexagonal, octagonal, etc.) and may passthrough the first (e.g., lower) plate, and into the reaction chamberwell. In some variations, the reaction chamber opening passes throughone or more actuation electrodes; in particular, the reaction chamberopening may be completely or partially surrounded by an actuationelectrode.

FIG. 1 shows a top view of an exemplary air-matrix DMF apparatus 101. Asshown, the DMF device may include a series of paths defined by actuationelectrodes. The actuation electrodes 103 are shown in FIG. 1 as a seriesof squares, each defining a unit cell. These actuation electrodes mayhave any appropriate shape and size, and are not limited to squares. Forexample, the unit cells formed by the actuation electrodes in the firstlayer may be round, hexagonal, triangular, rectangular, octagonal,parallelogram-shaped, etc. In the example of FIG. 1 , the squaresrepresenting the unit cells may indicate the physical location of theactuation electrodes in the DMF device or may indicate the area wherethe actuation electrode has an effect (e.g., an effective area such thatwhen a droplet is situated over the denoted area, the correspondingactuation electrode may affect the droplet's movement or other physicalproperty). The actuation electrodes 103 may be placed in any pattern. Insome examples, actuation electrodes may span the entire correspondingbottom or top surface the air gap of the DMF apparatus. The actuationelectrodes may be in electrical contact with starting sample chambers(not shown) as well as reagent chambers (not shown) for moving differentdroplets to different regions within the air gap to be mixed withreagent droplets or heated.

In the air-matrix apparatuses described herein, the first (lower) platemay also include one or more reaction chamber openings (access holes)105, 105′. Access to the reaction chamber wells may allow reactiondroplets to be initially introduced or for allowing reagent droplets tobe added later. In particular, one or more reaction droplets may bemanipulate in the air gap (moved, mixed, heated, etc.) and temporarilyor permanently moved out of the air gap and into a reaction chamber wellthough a reaction chamber opening. As shown, some of the reactionchamber openings 105′ pass through an actuation electrode. As will beshown in greater detail herein, the reaction chamber may itself includeadditional actuation electrodes that may be used to move a reactionchamber droplet into/out of the reaction chamber well. In somevariations one or more actuation electrodes may be continued (out of theplane of the air gap) into the reaction chamber well.

In general, one or more additional reagents may be subsequentlyintroduced either manually or by automated means in the air gap. In someinstances, the access holes may be actual access ports that may coupleto outside reservoirs of reagents or reaction components through tubingfor introducing additional reaction components or reagents at a latertime. As mentioned, the access holes (including reaction chamberopenings) may be located in close proximity to a DMF actuationelectrode(s). Access holes may also be disposed on the side or thebottom of the DMF apparatus. In general, the apparatus may include acontroller 110 for controlling operation of the actuation electrodes,including moving droplets into and/or out of reaction chambers. Thecontroller may be in electrical communication with the electrodes and itmay apply power in a controlled manner to coordinate movement ofdroplets within the air gap and into/out of the reaction chambers. Thecontroller may also be electrically connected to the one or moretemperature regulators (thermal regulators 120) to regulate temperaturein the thermal zones 115. One or more sensors (e.g., video sensors,electrical sensors, temperature sensors, etc.) may also be included (notshown) and may provide input to the controller which may use the inputfrom these one or more sensors to control motion and temperature.

As indicated above, surface fouling is an issue that has plaguedmicrofluidics, including DMF devices. Surface fouling occurs whencertain constituents of a reaction mixture irreversibly adsorbs onto asurface that the reaction mixture is in contact with. Surface foulingalso appears more prevalent in samples containing proteins and otherbiological molecules. Increases in temperature may also contribute tosurface fouling. The DMF apparatuses and methods described herein aim tominimize the effects of surface fouling. One such way is to perform thebulk of the reaction steps in a reaction chamber that is in fluidcommunication with the air gap layer. The reaction chamber may be aninsert that fits into an aperture of the DMF device as shown in FIGS. 2Band 2C. FIG. 2B shows the floor (e.g., first plate) of an air gap regioncoupled to a centrifuge (e.g., Eppendorf) tube 205 while FIG. 2Cincorporates a well-plate 207 (e.g., of a single or multi-well plate)into the floor of the air gap region. A built-in well 209 may also bespecifically fabricated to be included in the air-matrix DMF apparatusas shown in FIG. 2D. When a separate or separable tube or plate is used,the tubes may be coupled to the DMF device using any suitable couplingor bonding means (e.g., snap-fit, friction fit, threading, adhesive suchas glue, resin, etc., or the like).

In general, having a dedicated reaction chamber within the DMF deviceminimizes surface fouling especially when the reaction is heated. Thus,while surface fouling may still occur within the reaction chamber, itmay be mainly constrained to within the reaction chamber. This allowsthe majority of the air gap region floor to remain minimallycontaminated by surface fouling and clear for use in subsequent transferof reagents or additional reaction materials if needed, thus allowingfor multi-step or more complex reactions to be performed. When thereaction step or in some instances, the entire reaction is completed,the droplet containing the product may be moved out of the reactionchamber to be analyzed. In some examples, the product droplet may beanalyzed directly within the reaction chamber.

In order to bring the droplet(s) containing the starting materials andthe reagent droplets into the reaction chamber, additional actuationelectrodes, which may also be covered/coated with a dielectric and ahydrophobic layer (or a combined hydrophobic/dielectric layer), may beused. FIGS. 3A-3E shows a series of drawings depicting droplet 301movement into and out of an integrated well 305. As this series ofdrawings show, in addition to lining the floor of the air gap layer,additional actuation electrodes 307 line the sides and the bottom of thewell. In some variations, the same actuation electrode in the air gapmay be extended into the reaction chamber opening. The actuationelectrodes 307 (e.g., the reaction chamber actuation electrodes) may beembedded into or present on the sides and bottom of the well for drivingthe movement of the droplets into/out of the reaction chamber well.Actuation electrodes may also cover the opening of the reaction chamber.In FIG. 3A, a droplet 301 (e.g., reaction droplet) in the air gap layermay be moved (using DMF) to the reaction chamber opening. The actuationelectrodes 307 along the edge of the well and the sides of the wellmaintain contact with the droplet as it moved down the well walls to thebottom of the well (shown in FIGS. 3B and 3C). Once in the reactionchamber well, the droplet may be covered (as described in more detailbelow, either by placing a cover (e.g., lid, cap, etc.) over thereaction chamber opening and/or by mixing the droplet with a covering(e.g., encapsulating) material such as an oil or wax (e.g., when thedroplet is aqueous). In general, the droplet may be allowed to reactfurther within the well, and may be temperature-regulated (e.g., heated,cooled, etc.), additional material may be added (not shown) and/or itmay be observed (to detect reaction product). Alternatively oradditionally, the droplet may be moved out of the well using theactuation electrodes; if a mechanical cover (e.g., lid) has been used,it may be removed first. If an encapsulating material has been used itmay be left on.

In some variations contacts may penetrate the surfaces of the reactionchamber. For example, there may be at least ten electrical insertionpoints in order to provide sufficient electrical contact between theactuation electrodes and the interior of the reaction chamber. In otherexamples there may need to be at least 20, 30, or even 40 electricalinsertion points to provide sufficient contact for all the interiorsurfaces of the reaction chamber. The interior of the reaction chambermay be hydrophobic or hydrophilic (e.g., to assist in accepting thedroplet). As mentioned, an electrode (actuation electrode) may apply apotential to move the droplets into and/or out of the well.

In general, the actuation electrodes may bring the droplet into the wellin a controlled manner that minimizes dispersion of the droplet as it ismoved into the well and thus maintaining as cohesive a sample droplet aspossible. FIGS. 3D and 3E show the droplet being moved up the wall ofthe well and then out of the reaction chamber. This may be useful forperforming additional subsequent steps or for detecting or analyzing theproduct of interest within the droplet, although these steps may also oralternatively be performed within the well. Actuation electrodes may beon the bottom surface, the sides and the lip of the well in contact withthe air gap layer; some actuation electrodes may also or alternativelybe present on the upper (top) layer.

In instances where the reaction compartment is an independent structureintegrated with the DMF devices as those shown in FIGS. 2A and 2B, thethickness of the substrate (e.g., PCB) may be similar to what iscommonly used in DMF fabrication. When the reaction compartment is anintegrated well structure fabricated in the bottom plate of the DMFdevice as shown in FIG. 2D, the thickness of the substrate may beequivalent to the depth of the well.

In another embodiment, the electrodes embedded in the reactioncompartments can include electrodes for the electrical detection of thereaction outputs. Electrical detection methods include but are notlimited to electrochemistry. In some instances, using the changes inelectrical properties of the electrodes when the electrodes contact thereaction droplet, reagent droplet, or additional reaction component toobtain information about the reaction (e.g., changes in resistancecorrelated with position of a droplet).

The apparatuses described herein may also prevent evaporation.Evaporation may result in concentrating the reaction mixture, which maybe detrimental as a loss of reagents in the reaction mixture may alterthe concentration of the reaction mixture and result in mismatchedconcentration between the intermediate reaction droplet with subsequentaddition of other reaction materials of a given concentration. In somevariations, such as with enzymatic reactions, enzymes are highlysensitive to changes in reaction environment and loss of reagent mayalter the effectiveness of certain enzymes. Evaporation is especiallyproblematic when the reaction mixture has to be heated to above ambienttemperature for an extended period of time. In many instances,microfluidics and DMF devices utilizes an oil-matrix for performingbiochemical type reactions in microfluidic and DMF devices to addressunwanted evaporation. One major drawback of using an oil matrix in theDMF reaction is the added complexity of incorporating additionalstructures to contain the oil.

In general, described herein are methods and apparatuses for combatingevaporation by the use of wax (e.g., paraffin) in minimizing evaporationduring a reaction. A wax substance may include substances that arecomposed of long alkyl chains. Waxes are typically solids at ambienttemperatures and have a melting point of approximately 46° C. toapproximately 68° C. depending upon the amount of substitution withinthe hydrocarbon chain. However, low melting point paraffins can have amelting point as low as about 37° C., and some high melting point waxescan have melting points about 70-80° C. In some instances higher meltingpoint waxes may be purifying crude wax mixtures.

As mentioned, wax is one type of sealing material that may be used as acover (e.g., within a reaction chamber that is separate from the planeof the air gap). In some variations, wax may be used within the air gap.In particular, the wax may be beneficially kept solid until it isdesired to mix it with the reaction droplet so that it may coat andprotect the reaction droplet. Typically the wax material (or othercoating material) may be mixed with the reaction droplet and enclose(e.g., encapsulate, surround, etc.) the aqueous reaction droplet.

When a reaction droplet is maintained within a paraffin coating, notonly is evaporation minimized, but the paraffin may also insulate thereaction droplet from other potentially reaction interfering factors. Insome instances, a solid piece of paraffin or other wax substance may beplaced within a thermal zone of the air gap layer of the DMF device. Forexample, during a reaction, actuation electrodes may move a reactiondroplet to a wax (e.g., paraffin) body. Upon heating to a meltingtemperature, the wax body may melt and cover the reaction droplet. Thereaction then may continue for an extended period of time (including atelevated temperatures) without need to replenish the reaction solvents,while preventing loss by evaporation. For example wax-encapsulateddroplet may be held and/or moved to a thermal zone to control thetemperature. The temperature may be decreased or increased (allowingcontrol of the phase of the wax as well, as the wax is typically inertin the reactions being performed in the reaction droplet). Thetemperature at that particular thermal zone may be further increased tomelt the paraffin and release the reaction droplet. The reaction dropletmay be analyzed for the desired product when encapsulated by the liquidor solid wax, or it may be moved to another region of the DMF device forfurther reaction steps after removing it from the wax covering.Paraffins or other wax materials having the desired qualities (e.g.melting point above the reaction temperature) may be used. For example,paraffins typically have melting points between 50 and 70 degreesCelsius, but their melting points may be increased with increasinglonger and heavier alkanes.

FIG. 4A shows a time-sequence images (numbered 1-4) taken from anexample using a wax body within the air matrix as discussed above,showing profound reduction in evaporation as compared to a controlwithout wax (shown in FIG. 4B, images 1-2). In FIG. 4A, the first image,in the top right, shows an 8 μL reaction droplet 603 that has been movedby DMF in the air matrix apparatus to a thermal zone (“heating zone”)containing a solid wax body (e.g., paraffin wall 601). Once in position,the reaction droplet may be merged with a solid paraffin wall (e.g.,thermally printed onto DMF), as shown in image 2 of FIG. 4A, or the waxmaterial may be melted first (not shown). In FIG. 4A image 3, thethermal zone is heated (63° C.) to or above the melting point of the waxmaterial thereby melting the paraffin around the reaction droplet, andthe reaction droplet is surrounded/encapsulated by the wax material,thus preventing the droplet from evaporation as shown in FIG. 4A images3 and 4. Using this approach, in the example shown in FIG. 4A image 4,the volume of reaction droplets was maintained roughly constant at 63°C. for an incubation time approximately two hours long (120 min). Anequivalent experiment without the paraffin wall was performed, and shownin FIG. 4B. The left picture (image 1) in FIG. 4B shows the reactiondroplet 603′ at time zero at 63° C. and the right picture of FIG. 4Bshows the reaction droplet after 60 minutes at 63° C. As shown, thereaction droplet almost completely evaporated within approximately anhour's time at 63° C.

Through this approach of enclosing a droplet in a shell of liquid wax,the reaction volume and temperature are maintained constant without theuse of oil, a humidified chamber, off-chip heating, or dropletreplenishment methods. Waxes other than paraffin can be used to preventdroplet evaporation as long as their melting temperature is higher thanthe ambient temperature, but lower or equal to the reaction temperature.Examples of such waxes include paraffin, bees and palm waxes. Thewax-like solids can be thermally printed on the DMF device surface byscreen-, 2D- or 3D-printing. This wax-mediated evaporation preventionsolution is an important advancement in developing air-matrix DMFdevices for a wide variety of new high-impact applications.

As mentioned, the wax-based evaporation methods described may be used inconjunction with the DMF devices having a reaction chamber feature, orthey may be used without separate reaction chambers. When used within areaction chamber, the wax may be present in the reaction chamber and thereaction droplet may be moved to the reaction chamber containing wax forperforming the reaction steps requiring heating. Once the heating stephas completed, the reaction droplet may be removed from the reactionchamber for detection or to perform subsequent reaction steps within theair gap layer of the DMF device.

The methods and apparatuses described herein may be used for preventingevaporation in air-matrix DMF devices and may enable facile and reliableexecution of any chemistry protocols on DMF with the requirement for atemperature higher than the ambient temperature. Such protocols include,but are not limited to, DNA/RNA digestion/fragmentation, cDNA synthesis,PCR, RT-PCR, isothermal reactions (LAMP, rolling circleamplification-RCA, Strand Displacement Amplification-SDA, HelicaseDependent Amplification-HDA, Nicking Enzyme Amplification reaction-NEAR,Nucleic acid sequence-based amplification-NASBA, Single primerisothermal amplification-SPIA, cross-priming amplification-CPA,Polymerase Spiral Reaction-PSR, Rolling circle replication-RCR), as wellas ligation-based detection and amplification techniques (ligase chainreaction-LCR, ligation combined with reverse transcription polymerasechain reaction-RT PCR, ligation-mediated polymerase chainreaction-LMPCR, polymerase chain reaction/ligation detectionreaction-PCR/LDR, ligation-dependent polymerase chain reaction-LD-PCR,oligonucleotide ligation assay-OLA, ligation-during-amplification-LDA,ligation of padlock probes, open circle probes, and other circularizableprobes, and iterative gap ligation-IGL, ligase chain reaction-LCR, overa range of temperatures (37-100° C.) and incubation times (>2 hr).Additional protocols that can be executed using the systems and methodsdescribed herein include hybridization procedures such as for hybridcapture and target enrichment applications in library preparation fornew generation sequencing. For these types of applications,hybridization can last up to about 3 days (72 h). Other protocolsinclude end-repair, which can be done, for example, with some or acombination of the following enzymes: DNA Polymerase I, Large (Klenow)Fragment (active at 25° C. for 15 minutes), T4 DNA Polymerase (active at15° C. for 12 minutes), and T4 Polynucleotide Kinase (active at 37° C.for 30 minutes). Another protocol includes A-Tailing, which can be donewith some or a combination of the following enzymes: Taq Polymerase(active at 72° C. for 20 minutes), and Klenow Fragment (3′-5′ exo-)(active at 37° C. for 30 minutes). Yet another protocol is ligation byDNA or RNA ligases.

Manipulation and Processing of Encapsulated Droplets

Although the encapsulation of droplets in wax may prevent or reduceevaporation while executing chemistry protocols at elevatedtemperatures, after protocol completion, it has been discovered thatwhen the droplet is removed and separated from the wax, e.g., by drivingthe droplet using the electrodes of the DMF apparatus, a small amount ofliquid wax remains with the droplet as a coating even when the aqueousdroplet is moved away from the wax, and that this wax coating mayprevent or interfere with subsequent processing and analysis of thereaction droplet, particularly as the droplet cools and the waxsolidifies around the droplet after the droplet is moved out of theheating zone. Therefore, in some embodiments, the wax encapsulatedreaction droplet can be accessed through the wax coating using thesystems and methods described herein, which enables facile and reliableexecution of downstream biochemical processes.

To access the reaction droplet through the wax coating after thereaction droplet has been separated from the bulk liquid wax in theheating zone, an additional hydrophobic (e.g., oil) material may beadded to the reaction droplet to help dissolve the solidified waxencapsulated the reaction droplet. For example, a carrier droplet (i.e.,an aqueous droplet enclosed in a thin layer of oil) can be merged withthe encapsulated reaction droplet. The carrier droplet gains access tothe reaction droplet by having the oil from the carrier droplet dissolveand/or merge with the thin wax layer encapsulating the reaction droplet.Other materials other than oil may be used by the carrier droplet tobreak through the wax layer encapsulating the reaction droplet. Forexample, materials that are immiscible with aqueous reaction droplet andare capable of dissolving wax may be used, such as carbon tetrachloride,chloroform, cyclohexane, 1,2-dichloroethane, dichloromethane, diethylether, dimethyl formamide, ethyl acetate, heptane, hexane,methyl-tert-butyl ether, pentane, toluene, 2,2,4-trimethylpentane, andother organic solvents. Other materials that may be used to breakthrough the wax layer include ionic detergents such ascetyltrimethylammonium bromide, Sodium deoxycholate, n-lauroylsarcosinesodium salt, sodium n-dodecyl Sulfate, sodium taurochenodeoxycholic; andnon-ionic detergents such as dimethyldecylphosphine oxide (APO-10),dimethyldodecylphosphine oxide (APO-12), n-Dodecyl-13-D-maltoside(ULTROL®), n-dodecanoylsucrose, ELUGENT™ Detergent, GENAPOL® C-100,HECAMEG®, n-Heptyl (β-D-glucopyranoside, n-Hexyl-b-D-glucopyranoside,n-Nonyl-b-D-glucopyranoside, NP-40 Alternative, n-Octanoylsucrose,n-Octyl-b-D-glucopyranoside, n-Octyl-b-D-thioglucopyranoside, PLURONIC®F-127, Saponin, TRITON® X-100, TRITON® X-114, TWEEN® 20, TWEEN® 80,Tetronic 90R4. At temperatures where a wax remains liquid, a carrierdroplet encapsulated with wax may also be used to break through the waxencapsulating the reaction droplet. However, for lower temperatureswhere the wax solidifies, a carrier droplet coated with wax generallycannot be used since solid wax will prevent droplet movement.

For example, FIG. 7A illustrates a setup similar or the same as thatshown in FIG. 4A. The setup includes a DMF device interfaced to aheating element placed below or within the bottom DMF substrate, hencegenerating discrete heating zones 900 on the bottom DMF substrate.Alternatively, the heating element can be placed above or within the topsubstrate to form a heating zone on the top substrate. However, formingthe heating zone on the bottom substrate allows visual access. On thebottom substrate, a hydrophilic region 902 is printed or otherwiseformed or disposed around the actuating electrodes in the electrodearray 904 that are in the heating zone 900. One or more wax walls 906 orwax structures, which can be solid at room temperature, can be assembledon the top substrate by, for example, thermal printing to overlay aportion of the hydrophilic region 902 adjacent to the electrodes in theheating zone 900 on the bottom plate when the DMF device is assembled.Alternatively, the wax walls 906 or wax structures can be formeddirectly on the bottom plate around the electrodes in the heating zone900. In yet another embodiment, the wax walls 906 can be placed on aremovable sheet that can be removably attached to either the top plateor the bottom plate. The removable sheet can have a hydrophobic surfaceon one side for interacting with the droplet and an adhesive on theother side for adhering to the top or bottom plate. Reagents and othermaterials can also be placed on the removable sheet to interact with thedroplets. In some embodiments, the top plate or the bottom plate can bepart of a removable cartridge that is combined with the other plate andelectronics to form the working DMF device. As described herein, areaction droplet 908 can be transported to the heating zone 900 along apath of actuating electrodes, which may be a relatively narrow pathformed by a single line of actuating electrodes to the heating zone 900.Then the heating zone 900 is heated, and the wax wall 906 surroundingthe heating zone 900 and reaction droplet 908 melts to encapsulate thereaction droplet 908 in liquid wax 910 as shown in FIG. 7B (frame i),thereby preventing or reducing evaporation from the reaction droplet 908during the reaction protocol. The hydrophilic region 902 surrounding theheating zone 900 functions to pin or localize the liquid wax 910 inplace in the heating zone 900 and allows the reaction droplet 908 tobreak away as described below.

As shown in FIG. 7B (frames ii-iv), the process of breaking away orseparating the encapsulated reaction droplet 908 from liquid wax 910 canbe accomplished by driving the aqueous reaction droplet 908 away fromthe heating zone 900 and the liquid wax 910 by actuating the actuatingelectrodes in the heating zone and path. As the aqueous reaction droplet908 is actuated away from the heating zone 900, the hydrophilic region902 surrounding the liquid wax 910 helps hold the liquid wax 910 inplace as the reaction droplet 908 moves away from the heating zone 900,which causes the liquid wax 910 encasing the droplet 908 to begin toneck and eventually break off from the droplet 908, thereby leavingtrace or small quantities of liquid wax 910 surrounding the separatedreaction droplet 908. In general, the heating zone 900 is single useonly to avoid cross-contamination. However, in situations wherecross-contamination is not an issue, the heating zone 900 may be reusedby heating and melting the wax within the heating zone and then movingthe next droplet into the reheated liquid wax 910.

Because the reaction droplet may be surrounded by a thin layer of liquidwax 910 after separation from the heating zone 900, it may be difficultto merge the reaction droplet 908 with another aqueous droplet since theliquid wax 910 coating may act as a barrier. In addition, the liquid wax910 may solidify as the droplet cools to form a physical barrier thatimpedes merger with another droplet. Therefore, to facilitate merging ofa liquid wax 910 coated reaction droplet 908 or a cooled reactiondroplet 908 with a solid wax coating with another droplet, a carrierdroplet 912 can be used to merge with the reaction droplet 908 as shownin FIG. 7B (frame v). The carrier droplet 912 can be an aqueous dropletthat is coated with a thin layer of oil or another organic solvent asdescribed above. The aqueous portion of the carrier droplet 912 caninclude additional reagents, beads coated (or not) with DNA/RNA probesor antibodies or antigens for performing separations, uncoated beads,magnetic beads, beads coated with a binding moiety, solid phasereversible immobilization (SPRI) beads, water for dilution of thereaction droplet, enzymes or other proteins, nanopores, wash buffers,ethanol or other alcohols, formamide, detergents, and/or other moietiesfor facilitating further processing of the reaction droplet 908. Asshown in FIG. 8A (frames i-iv), when the carrier droplet 912 and thereaction droplet 908 are moved by the actuating electrodes to the samelocation, the thin layer of oil surrounding the carrier droplet 912 canmerge with the thin layer of liquid wax surrounding the reaction droplet908, thereby facilitating the merger of the aqueous portions of the twodroplets 908, 912 to form a combined droplet 914.

After the carrier droplet 912 has been merged with the reaction droplet908, further processing of the combined droplet 914 can proceed, such asextracting an analyte from the combined droplet 914 and/or perform othersteps such as hybridizing capture probes, digesting the reaction productusing an enzyme, amplifying the reaction product with a set of primers,and the like. For example, the carrier droplet 912 can be carrying beadsfor extracting the analyte, e.g., DNA or RNA or proteins. When thedroplets are merged, the beads, which can be magnetic, can be used tomix the combined droplet 914 by application of a magnetic field. Thetarget analyte binds to the beads, which can be immobilized against thesubstrate by the magnetic field to form a bead pellet 916, as shown inFIG. 8B (frame i). Next, the combined droplet 914 can be moved away fromthe immobilized bead pellet 916, leaving the bead pellet 916 with boundanalyte on the substrate, as shown in FIG. 8B (frames ii-iii). Thecombined droplet 914 can be moved away from the immobilized bead pellet916 by actuating the electrodes. Alternatively, the combined droplet 914can be held in place while the bead pellet 916 is moved away from thecombined droplet 914. The bead pellet 916 can be moved away andseparated from the combined droplet 914 by, for example, moving themagnetic field (e.g., by moving the magnet generating the magneticfield) that is engaging the bead pellet 916 away from the combineddroplet 914. In some embodiments, the combined droplet 914 can beactively immobilized through actuation of the electrodes in contact withthe droplet and/or surrounding the droplet. Alternatively or inaddition, the droplet 914 can be passively immobilized through naturaladhesive forces between the droplet and substrate on which the dropletis contacting, as well as physical structures, such as retaining wallsthat partially surround the combined droplet 914 while having an openingfor passing the bead pellet 916. As shown in FIG. 8C (frames i and ii),an aqueous droplet 918 can be moved over the bead pellet 916 toresuspend the beads with the bound analyte. See Example 3 describedbelow for an embodiment of this procedure used for miRNA purification.

Example 1: Device Fabrication and Assembly

DMF apparatuses that include embedded centrifuge tubes and/or well-platewells (e.g., FIGS. 2B, 2C) were constructed by drilling 5.5 mm diameterholes into 3 mm thick PCB substrates, bearing copper (43 μm thick)plated with nickel (185 μm) and gold (3.6 μm) for electrodes andconductive traces. Tubes and wells were then inserted into holes. DMFdevices with embedded wells (e.g., FIG. 2D) were fabricated with holes(5 mm diameter, 10 mm depth) drilled in 15 mm thick PCB substrates.Actuation electrodes (each 10 mm×10 mm) were formed by conventionalphotolithography and etching, and were coated with soldermask (˜15 μm)as the dielectric. As shown in FIGS. 3A-3E, some of the electrodes wereformed around and adjacent to the hole which served as the access pointto reaction compartments. The electrical contact pads were masked withpolyimide tape (DuPont; Hayward, Calif.), and the substrate wasspin-coated with a 50 nm layer of Teflon-AF (1% wt/wt in FluorinertFC-40, 1500 rpm for 30 sec) and then baked at 100° C. for 3 h. The topplate of the DMF device, consisting of a glass substrate coateduniformly with unpatterned indium tin oxide (ITO) (Delta TechnologiesLtd; Stillwater, Minn.) with 5.5 mm diameter PDMS plugs was spin-coatedwith 50 nm of Teflon-AF, as described above.

Prototype devices fabricated as described above performed better or aswell as air-gap DMF apparatuses without reaction chambers.

Example 2: Quantifying Evaporation Prevention Using Waxes

To qualitatively evaluate the effect of wax bodies to preventevaporation in our assays, loop mediated amplification (LAMP) reactionswere executed while covered in liquid paraffin wax in tubes on thebenchtop using a real-time PCR Machine. As shown in FIG. 5 , the LAMPassay amplified miR-451, and the Ct values with and without paraffinwere comparable (˜13 cycles), indicating no significant effect on theassay. For LAMP on DMF, the reaction droplet (8 μL) was driven toheating zone (as shown in FIG. 4A). There, the droplet wets the solidparaffin wax wall which under conditional heating at 63° C. will meltinto liquid wax to encircle the reaction volume and maintain it intactthroughout the incubation time at 63° C. FIG. 6A shows a LAMP assayusing paraffin-mediated methods, while FIG. 6B shows a LAMP assay usingconventional methods. In FIG. 6A, the two upper traces are for ahemolyzed sampled while the two lower traces are for a non-hemolyzedsample. The two traces of each are to show repeatability of the runsusing wax-mediated air matrix DMF. In FIG. 6B, the conventional LAMPassay for a hemolyzed sample are shown in upper two traces while thenon-hemolyzed LAMP runs are shown in lower two traces. Again, the twoupper and two lower traces each are to show result repeatability. Thewax-mediated approach on DMF generated results comparable in Ct valuesto those generated by conventional LAMP in tubes as shown in FIGS. 6Aand 6B.

Example 3: miRNA Purification

Human Panel A beads from the TaqMan® miRNA ABC Purification Kit (ThermoFisher Scientific). Aliquots of miRNA (4 ul), or “reaction droplets”,were loaded onto the DMF platform and brought to an array of electrodesoverlaying the heating zone such that the droplet came into contact withthe paraffin wall. The heating zone was then heated (65° C., 2 min) tomelt the paraffin around the droplet. Once the paraffin melted, thereaction droplets were driven away from the heating zone and merged withmiRNA Binding Beads (4×106 beads; FIG. 3A) in 2 ul of mineral oil (i.e.,carrier droplet). After mixing, the droplets were incubated (30° C., 30min) to allow miRNA to bind to the miRNA Binding Beads. Beads werecaptured by engaging an external magnet positioned below the bottomplate. Once a pellet was formed, the beads were recovered from solutionby moving the magnet laterally along the bottom plate whilesimultaneously actuating the electrodes positioned below the reactiondroplet (FIG. 3B). The miRNA Binding Beads were then resuspended inwater (4 ul) using the DMF platform and transferred to a centrifuge tubefor elution of miRNA (70° C., 3 min; FIG. 3C). The efficiency of miRNArecovery from paraffin-encased miRNA droplets was evaluated againstrecovery from miRNA droplets without paraffin, but only in oil. RT-qPCRanalysis of miRNA prepared by the system from samples with and withoutparaffin encasement generated comparable Ct values.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. A method of performing droplet operations on adroplet at least partially coated in liquid wax within an air-matrixdigital microfluidic (DMF) apparatus, the method comprising: moving, byelectrowetting, an aqueous reaction droplet having an outer coating ofliquid wax within an air gap of the air-matrix DMF apparatus which isformed between a first plate and a second plate of the air-matrix DMFapparatus; merging the aqueous reaction droplet with a carrier dropletcomprising an aqueous droplet coated with an oil or an organic solventin the air gap to form a combined droplet; and moving, byelectrowetting, the combined droplet within the air gap.
 2. The methodof claim 1, wherein moving, by electrowetting, the aqueous reactiondroplet comprises initially transporting the aqueous reaction droplet toa thermal zone of the air gap, the thermal zone comprising a wax andmelting the wax within the thermal zone to at least partiallyencapsulate the aqueous reaction droplet with the wax.
 3. The method ofclaim 2, further comprising regulating a temperature of the at leastpartially encapsulated reaction droplet to allow a reaction to proceedwithin the aqueous reaction droplet before transporting the aqueousreaction droplet having the outer coating of liquid wax within the airgap.
 4. The method of claim 2, wherein melting the wax comprises meltinga solid wax formed into a wall or open chamber within the air gap. 5.The method of claim 1, wherein moving, by electrowetting, the aqueousreaction droplet comprises transferring the aqueous reaction dropletaway from away from a thermal zone comprising a wax material so that atleast some of the wax is left behind.
 6. The method of claim 1, furthercomprising detecting a product within the aqueous reaction droplet orthe combined droplet.
 7. The method of claim 1, wherein merging theaqueous reaction droplet with the carrier droplet comprises moving oneor both of the aqueous reaction droplet and the carrier droplet intocontact with each other by electrowetting.
 8. The method of claim 1,further comprising mixing the combined droplet, wherein the combineddroplet comprises a plurality of beads.
 9. The method of claim 8,further comprising immobilizing the beads.
 10. The method of claim 9,further comprising moving the combined droplet away from the immobilizedbeads.
 11. The method of claim 10, further comprising re-suspending theimmobilized beads within an aqueous droplet.
 12. The method of claim 8,further comprising separating the beads from the combined droplet bymoving a magnetic field away from the combined droplet to magneticallydraw the beads away from the combined droplet.